This invention relates to a non-contacting apparatus and method for investigating certain properties of blood such as red cell count, haemoglobin and fibrinogen content, sedimentation rate and related physical and chemical parameters and for use with similar evaluations in other biological media and for more general use with other samples. In one example the invention is concerned with measurement of fibrinogen to establish instantly the expected sedimentation condition of red cells in blood and certain plasma properties.
Throughout, the term "non-contacting" indicates a means remote from and not in direct contact with the sample and the terms "instant" and "instantaneous" are used to indicate very near instant with process times only being limited by the speed of electron flow in circuitry, real time electronic calculation and the operation time of electronic display devices.
Protein concentration in biological media is usually assessed by biochemical methods or by methods of physical chemistry such as viscosity measurement and optical rotational dichroism. Also possible arc various forms of spectroscopic analysis and chromatography. In one specific situation, that of whole blood, the proteins with the highest concentration are haemoglobin, found in the erythrocyte nuclei and secondly fibrinogen, found dissolved in the plasma. Fibrinogen concentration is medically important, in that in excess it is a non-specific indicator of disease state in a person. Fibrinogen levels manifest their effects in a variety of different ways; firstly, they effect the sedimentation rate of the red cells (erythrocytes) giving rise to the so-called erythrocyte sedimentation rate (ESR) and secondly, they cause effect upon the physical and chemical properties of the plasma.
Manifestations of increased fibrinogen levels have traditionally been monitored in pathology laboratories by two tests, namely; the ESR and the plasma viscosity (PV); more recently a third biochemical assay, the so-called c-reactive protein (CRP) test has also become more popular. ESR tests are, however still the most popular with clinicians the world over. The ESR test traditionally uses about 5 milli-liters of venous blood and takes one hour to perform, during which time the red cell fraction (haematocrit) separates from the clearer plasma fraction and sediments slowly under gravity and subject to internal viscoelastic forces down a capillary tube or a. vacutainer containing preservative; this is very time-consuming. PV and CRP are also time-consuming and, because in these latter two tests, the red and white blood fractions have to be physically or chemically separate, there is always the chance, albeit remote, that the operatives might become exposed to viral or bacterial biohazard.
Other blood tests such as cell counting and sizing are also carried out in pathology laboratories using very expensive automated equipment which needs to sample small quantities of blood in close contact by sucking it through a needle type probe inserted by the equipment into a closed vacutainer. Such cell counters, sometimes referred to as haematological analysers, Coulter or similar cell counters, are extremely sophisticated and operate by application of non-linear electrical field gradients and voltage pulses across individual red or white blood cells which have been located by electric or hydrodynamic focusing in a narrow, micron sized, orifice or counting/sizing gate. These machines yield a myriad of parameters, up to 23 in some cases, about the state of nearly all the blood components. Nevertheless, they are non-portable and extremely expensive and limited by sample throughput and cleansing procedures.
Three of the most important parameters outputted by cell counters are perhaps the rod cell concentration (RBC), the mean cell volume (MCV) and the haemoglobin content (Hb). These parameters are considered very useful by many physidans in addition to the ESR value in order to make first diagnoses and general "state of health" assessments, and it would be useful if such parameters could be obtained by a simpler, cheaper, haematology or haematological analyser technology of greater portability, for use, for example, in medical practitioners offices, in the field, or in connection with third world applications.
Of these parameters, the problem of haemoglobin has been addressed by using optical technology and biochemical analysis of the erythrocytes. However, such technology is still quite expensive and because a chemical, reaction is involved there is a waiting time before the result is achieved, i.e. the output is not instantaneous.
Most matter is electrically and magnetically permeable to varying extends. The property, which influences electric fields or the electric field vector of electromagnetic waves (radiation) is the permittivity or relative dielectric constant .epsilon., while that with magnetic effect is the (magnetic) permeability .mu.. Generally there are many more materials of low .mu. end a range of .epsilon., the so-called dielectrics, than there are magnetic materials of high .mu..
In dielectric laboratories, electrical properties of matter, especially liquids, are measured by bridge apparatus in which linear a.c. electric fields are employed end direct contact with metal electrodes is usually made. Alternatively, time domain pulse reflectometry is employed where the sample is housed in a metal cavity to form the termination impedance of a coaxial feed-line. These are standard techniques of so-called dielectric measurement.
The parameters obtained by such measurement are the frequency dependent dielectric parameters e', e" and tan .delta.. These are often obtained for the sake of pure scientific research. Alternatively they may be mathematically or empirically related to, or are indeed characterized by, the physical and/or chemical state/properties of the sample. For instance, if the sample is a liquid containing particles in suspension, the size and number density of these may be hypothetically related to the dielectric parameters.
The haematological parameters of blood are manifold and complicated, in brief being related to the chemical and biochemical composition of the blood electrolytes and plasma and to the sizes and number densities of red and white cells and to the electrical charge states of their membrane surfaces and walls. Properties of blood and other cells are traditionally determined by Coulter apparatus in which individual cells are manipulated into a counting/sizing dimension, manipulation and measurement often involving hydrodynamic focusing and the application of pulsed non-linear electric field gradients.
Coulter apparatus is extremely expensive, yet because of medical demand, is widely exploited. On the other hand, a system not in current commercial exploitation involves dielectric measurement of pathological blood samples to yield haematological parameters. Although academics have attempted to assess the dielectric properties of blood in the laboratory, it would seem, according to scientific literature that they have always employed pooled samples of cells of various mammalian species separated into individual red and white fractions, often suspended in artificial electrolyte media and always in contact with metal and employing the standard techniques described above.
Although there is a moderate amount of scientific literature on this type of approach, there is no really consistent agreement on the observed relaxation frequencies of mechanisms pertaining thereto. Indeed, serious errors of measurement can be introduced in test vessels where a liquid sample is contained in contact with metal for the purpose of dielectric measurement due to electrical double layer formation and electrode polarisation at the liquid metal interface and due to chemical reaction with aggressive chemical media, such as whole blood or blood fractions.
Recently, some new but relatively simple methods have been described for applying fixed frequency non-contacting dielectric (capacitive) measurements to flowing solids, e.g. fly-ash, as in UK patent application GB 2,115,933A, published on 14 September 1983. Similar techniques with vertically positioned, essentially parallel external electrodes forming part of a resonant LC circuit have been applied to the ease of flowing fluids, e.g. European patent application EP 0,309,085 A2, published 29 March 1989. However, such simple capacitive techniques effectively only measure the electrical permittivity in simple form, and only at a single frequency and not in complex frequency dependent form as referred to above, where: EQU .epsilon."(.omega.)=.epsilon.'(.omega.)-j.epsilon."(.omega.)
Because of this complex form, single frequency methods are strongly influenced by factors such as the D.C. ionic conductivity of liquid samples and/or the position(s) in frequency space, relative to the measurement frequency, of the dielectric loss maximum or maxima, and thus are not wholly satisfactory, particularly if a sample exhibits a multiple dielectric dispersion, of which blood is one such example.
It has also been recently recognised that the flow of electromagnetically permeable samples in tubes may be monitored by wrapping a non-contacting inductor around the tube and connecting it to essentially free running oscillator circuits so that either the self-resonant frequency of the inductor or resonant frequency of the inductor in series or parallel combination with a capacitor, crudely determines the oscillation frequency of the oscillator.
Alternatively, the coil may be driven with a.c. voltage in the region of parallel resonance and effective measurement of the Q-curve obtained by measuring the voltage across the coil. For dielectric samples, this procedure effectively measures non-complex, single frequency permittivity by its effect of dielectric loading and lowering of the Q of the coil, thus the above restrictions of two capacitor plate methods also apply. These very restrictions themselves, particularly with respect to low frequency dispersions or d.c. conductivity involving ionic conduction, and make the use of these methods suitable for some purposes such as detection of ion concentration in a liquid; see U.S. Pat. No. 4,590,424.
Similar inductive techniques have been applied to flowing particles where the predominant change in the coil is that of inductance rather than Q, the former being brought about by the particles significant magnetic permeability rather than simply dielectric constant alone. An example is European patent application 0,157,496 A2, published 9 October 1985. Whilst these wholly free running methods of oscillation are adequate for the purposes for which they have been employed they are not stable enough or sensitive enough for haematological purposes.
The material which is of prime concern for the present invention is blood, which has the potential to behave both as a dielectric and be magnetically permeable via the iron in the haemoglobin molecule. GR 1,574,681, granted to Labora Mannheim and published 10 September 1980 concerns one haematological aspect, namely time-dependent erythrocyte sedimentation. It employs either two electrode capacitive techniques or inductive LC techniques. Although a retuning mechanism is employed in assessing the sedimentation rate, the technique is still essentially a single frequency one, or at least the device operates within a single relatively narrow band of resonance frequencies about some mean which is not explicitly specified, and requires observations to be made over a period of time.
As with academic literature, patent literature seems to indicate that no attempts have been made to apply non-contacting dielectric methods for commercial exploitation in the haematological field for determination of standard haematological parameters other than time-dependent erythrocyte sedimentation rate. The other common hitherto dielectrically unexploited parameters include cell number density, size and haemoglobin concentration, usually referred to as RBC, MCV and Hb respectively. The present invention suggests that the restrictions on previous exploitations could be due to the general limitations of single two plate capacitor and single inductor methods as outlined above, particularly with respect to finite variations found with pathological blood specimens in plasma and electrolyte conductivity and due to the multiple dielectric loss mechanisms and maxima to be expected as arising from blood cell size, shape and molecular motions of the haemoglobin molecule and other protein and other molecules present in such pathological blood samples. It will be appreciated by those with knowledge in these fields that other fluids and materials both in vitro and in vivo could fall into this "restricted" category, i.e. subject to the same or similar limitations.